Solution phase electrophoresis device, components, and methods

ABSTRACT

A device for fluid phase electrophoresis, particularly solution phase isoelectric focusing, components thereof, and methods for use are presented.

FIELD OF THE INVENTION

The present invention is in the field of devices, components, andmethods for electrophoresis, particularly isoelectric focusing, moreparticularly solution phase isoelectric focusing (IEF).

BACKGROUND OF THE INVENTION

The complexity of eukaryotic proteomes—that is, the total number ofdistinct protein species present concurrently in a eukaryotic cell,including alternatively spliced isoforms and variants differing inpost-translational modification—typically exceeds the resolving capacityof current analytical techniques.

For example, the number of distinct protein species in eukaryotic cellstypically far exceeds the spatial resolution of two-dimensionalpolyacrylamide gel electrophoresis (2D PAGE) gels, with large numbers ofdistinct protein species appearing to comigrate. The limited spatialresolution in turn constrains the dynamic detection range of thetechnique: efforts to observe low abundance species by increasing theinitial protein load lead to increased obscuration by high abundancespecies.

Recently, efforts have been made to increase resolution of such proteinanalytical techniques by prefractionating the protein mixture prior toanalysis. In one approach, complex mixtures are prefractionated usingsolution phase isoelectric focusing, yielding fractions having distinctpI ranges that can thereafter be separately analyzed by 2D PAGE withincreased resolution. See Zuo and Speicher, Anal. Biochem. 284:266-278(2000); Zuo et al., Electrophoresis 22:1603-1615 (2001); Zuo andSpeicher, Proteomics 2:58-68 (2002); Ali-Khan et al., Current Protocolsin Protein Science 22.1:1-19 (2002); Zuo et al., J. Chromatography B782:253-265 (2002); and Wistar Institute, WO 01/75432, the disclosuresof which are incorporated herein by reference in their entireties. Seealso Tan et al., Electrophoresis 23:3599-3607 (2002); WO 01/36449; WO00/17631; and Righetti et al., J. Chromatography 475:293-309 (1989).

A number of devices capable of solution phase isoelectric focusing areavailable commercially. None of the devices, however, providesparticularly convenient solution phase IEF prefractionation of smallvolume protein samples with a simple device in a format that readilyinterfaces with subsequent analytical techniques such as 2D PAGE.

There thus exists a continuing need in the art for devices, components,and methods for solution phase electrophoresis, particularly solutionphase isoelectric focusing.

SUMMARY OF THE INVENTION

The present invention satisfies these and other needs in the art byproviding a solution phase isoelectric focusing device that is assembledby simply inserting components (e.g., chambers) into a loading tube andholding them in place with a single screw cap. The screw cap istightened to ensure leak-proof seals between the chambers and othercomponent parts. The device is easy to disassemble by simply sliding thechambers and other components out of the loading tube. In preferredembodiments, air bubbles are avoided in sample chambers by usingoptional specially shaped openings and cap seals to seal the chambersafter loading and/or during electrophoresis or subsequent manipulations.

Generally, in IEF applications, the device uses fixed pH membrane disks.Such disks can have a shape that limits the electrodecantation ofproteins. The shape of the membrane disks can also be used to properlyorientate a disk in position between two chambers, thus minimizing oreliminating leaks that arise from poorly orientated disks. Disk shapesthat have these and other desirable aspects are disclosed herein.

In one aspect, the invention relates to devices for IEF. In someembodiments, the device does not require means for sample recirculation,mixing or other agitation within its chambers during the electrophoreticseparation. In other embodiments, however, the device is positioned on arocker platform or a rotary platform to move the entire device duringoperation, thus effecting sample agitation.

In another embodiment, the invention relates to IEF devices having aloading tube into which a plurality of sample chambers is loaded. Thetube aligns the sample chambers in a coaxial orientation during assemblyand operation. The chambers may be compressed using one or moreactuators, such as a screw cap at one end of the loading tube.

Thus, in some embodiments, sample chambers are held tightly in positionby a screw cap at one end of the loading tube. The screw cap compressesthe chambers against one another as it is tightened. Sealing O-ringsbetween the chambers compress under the pressure to form leak-proofseals.

In further embodiments, an anode end piece is introduced to the loadingtube before the first sample chamber to facilitate subsequentdisassembly of the device. The anode end piece has a protrusion thatslides along a channel in the loading tube, facilitating removal ofchambers from the loading tube.

In yet further embodiments, the electrode wires, which are submerged inelectrode buffer during use, are housed in circumferential detents incylindrical electrode plugs. The recessed electrode wires are protectedfrom damage during use and during cleaning. The electrode plugs areoptionally removable from the device for cleaning, repair orreplacement. Preferably, the electrodes provide for a more consistentelectrical field regardless of support structure rotational orientation.

In yet further embodiments, the sample chambers have loading ports andcorresponding cap seals having a design such that air is not trapped insample chambers when they are sealed. A cap seal of the invention doesnot completely block the fill port until the cap seal is fully inserted,so that no air is trapped in closing the sample chambers.

Moreover, the present invention relates generally to methods and designsfor sealing sample chambers without trapping air bubbles, wherein air isdisplaced from sample chambers by slightly over-filling the samplechamber with a supernatant fluid, and the excess of such fluid isdisplaced as the cap seal is inserted into the loading port. Suchmethods and cap seals can be used in other applications, both in thoseinvolving aqueous solutions, as well as other liquids. Other liquidsinclude without limitation organic solvents (e.g., benzene, otheraromatic hydrocarbons, chlorinated hydrocarbons, hexane,dichloromethane, alcohols, ketones, ethers, amines, esters, petroleumproducts (oil, gas, etc.), paints, primers, sealants, liquefied gases(e.g., liquid oxygen, liquid nitrogen, etc.), culture media (i.e., foranaerobic bacteria) and the like. In general, this aspect of theinvention applies to any liquid container the use of which would beenhanced by reduced contact with air and/or fewer air bubbles.

In another aspect, the present invention relates to immobilized buffermembranes for use in isoelectric focusing in one or more otherembodiments of the invention.

In a further aspect, the invention relates to kits for solution IEFcomprising the disks of the invention. Optionally, such kits furthercomprise one or more buffers, one or more sets of instruction, one ormore protein standards, and/or one or more control samples.

In a further aspect, the invention relates to methods of separating,fractionating and characterizing proteins and other biomolecules using adevice of the invention. Such processes involve electrophoresis,including without limitation isoelectric focusing (IEF).

BRIEF DESCRIPTION OF THE DRAWINGS

These and other objects and advantages of the present invention will beapparent upon consideration of the following detailed description, takenin conjunction with the accompanying drawings, in which like graphicalrepresentations and like characters refer to like structures throughout,and in which the terms “proximal” and “distal” refer to the position ofa component relative to the anode when the device of the presentinvention is assembled and ready for use.

FIG. 1 is a partially exploded proximal perspective view (from above) ofone embodiment of the device of the present invention.

FIG. 2A is a partially exploded proximal perspective view (from above)of one embodiment of the device of the present invention, without thelid shown in FIG. 1.

FIG. 2B is a proximal perspective view (from above) of the componentsshown in FIG. 2A, fully assembled. The axis of the electrical field isindicated by a dashed line.

FIG. 3A is an exploded proximal perspective view (from above) of thecathode buffer chamber and the spill trough of an embodiment of thedevice of the present invention, with the cathode electrode plugdisassembled from the cathode buffer chamber.

FIG. 3B is a proximal perspective view (from above) of the cathodebuffer chamber and the spill trough of an embodiment of the device ofthe present invention, with the cathode electrode plug operationallyengaged in the cathode port.

FIG. 4A is a distal perspective view (from above) of the anode bufferchamber and the loading tube of an embodiment of the device of thepresent invention, with the anode electrode plug disassembled from theanode port.

FIG. 4B is a distal perspective view (from above) of the anode bufferchamber and the loading tube of an embodiment of the device of thepresent invention, with the anode electrode plug operationally engagedin the anode buffer chamber.

FIG. 5A is a partially exploded proximal perspective view (from above)of the anode buffer chamber, the loading tube, and the chamber stack ofan embodiment of the present invention. In this exemplary view, sealingO-ring 29, immobilized buffer disk (IBD) 28, and spacer 30 are all shownpositioned between two sample chambers 2; in typical embodiments,however, IBD 28 and spacer 30 are alternatives, and only one of the twois positioned with an O-ring between adjacent sample chambers.

FIG. 5B is a partially exploded distal perspective view (from above) ofthe same components as in FIG. 5A.

FIG. 5C is a distal perspective view (from above) of the anode bufferchamber, the loading tube, and several sample chambers and associatedcomponents of an embodiment of the present invention, assembled, butwithout the screw cap.

FIG. 5D is a distal perspective view (from above) of the componentsshown in FIG. 5C, with the screw cap operationally engaged.

FIG. 6A is an exploded proximal perspective view (from above) of oneembodiment of a sample chamber of the present invention, with fill port,a cap seal for the fill port, a sealing O-ring, and a spacer.

FIG. 6B is an exploded distal perspective view (from above) of oneembodiment of a sample chamber according to the present invention,showing the use of an IBD as an alternative to use of a spacer.

FIG. 6C is a proximal perspective view (from above) of an embodiment ofa sample chamber of the present invention showing a cap seal engaged inthe chamber fill port, and a sealing O-ring, assembled.

FIG. 7A is an orthogonal midsectional view of an embodiment of a samplechamber of the present invention, viewed along line A-A of FIG. 6C,showing the fill port.

FIG. 7B is an orthogonal midsectional view of the sample chamber of FIG.7A, showing a cap seal operationally inserted into the fill port.

FIG. 7C is an orthogonal midsectional view of an alternative embodimentof a sample chamber of the present invention having a tapered fill port,viewed along line A-A of FIG. 6C.

FIG. 7D is an orthogonal midsectional view of the sample chamber of FIG.7C, showing an alternative cap seal, properly dimensioned to seal thetapered fill port, engaged within the fill port.

FIG. 7E is a bottom orthogonal view of an embodiment of the cap seal ofFIG. 7D.

FIG. 8 is a distal perspective view (from above) of an embodiment of acathode end piece of the present invention; the end piece interfaces thedistal-most sample chamber of the chamber stack with the screw cap (notshown).

FIG. 9A is a proximal perspective view (from above) of a lid andassociated anode and cathode cables, according to one embodiment of thepresent invention. Also shown are the anode and cathode tab slots of thelid.

FIG. 9B is a proximal perspective view (from above) of the lid andassociated electrode cables of FIG. 9A, assembled with additionalcomponents of an embodiment of a device according to the presentinvention. In the embodiment shown, the cathode tab slot of the lid isshaped to receive only the cathode tab of the device, thus preventingthe lid from being assembled with the device except in the orientationshown.

FIG. 10A is a partial side midsectional view of several sample chambersassembled into a chamber stack, according to one embodiment of thepresent invention, exploded to show the cap seals positioned forinsertion.

FIG. 10B shows the cap seals engaged within the sample chambers of FIG.10A.

FIG. 11A is a front (or equivalently, back) orthogonal view of anexemplary immobilized buffer disk (IBD) of the present invention havingan ovoid (pseudoelliptical) shape. FIG. 11B gives an exemplary formulafor designing a disk with such shape.

FIGS. 12A-12F are scanned images of 2D gels—obtained by immobilized pHgradient (IPG) isoelectric focusing, using either pH 3-10 strips, pH 4-7strips, or pH 6-10 strips, as indicated immediately below each gelimage, followed by SDS-PAGE—demonstrating the effects ofprefractionating a rat liver tissue lysate into five separate fractionsusing a device, components, and methods of the present invention. FIG.12A is obtained with unfractionated lysate. Each of FIGS. 12B-12F isobtained using a fraction from a different one of the device samplechambers; the pH range of the device sample chamber is shown in largetype below the IPG strip pH range.

FIGS. 13A-13E are scanned images of 2D gels. FIG. 13A is obtained fromthe pI 4.6-5.4 lysate fraction using a pH 4-7 IPG strip, FIG. 13B fromthe pI 4.6-5.4 lysate fraction using a pH 4.5-5.5 narrow range IPGstrip, with FIG. 13C showing an enlargement of the indicated region ofthe gel shown in FIG. 13B. FIG. 13D is obtained from unfractionated ratliver lysate using a pH 4.5-5.5 IPG strip, with FIG. 13E showing anenlargement of the indicated region of the gel shown in FIG. 13D.

FIGS. 14A and 14B show an exemplary embodiment of a cathode plug of thepresent invention. FIG. 14A is a top perspective schematic view of theplug, without the electrode wire, particularly indicating the outlet fortraversal of the wire from the plug interior to its exterior, and thecircumferential detent near the plug bottom, around which the electrodewire wraps. FIG. 14B shows the plug of FIG. 14A with the electrode wirepassing from the electrode, through the plug interior, through theoutlet, and around the circumferential detent.

DETAILED DESCRIPTION OF THE INVENTION

In a first aspect, the invention provides a device, and componentsthereof, for solution phase electrophoretic separation of analyteswithin a sample.

The device comprises an anode, a cathode, a chamber stack disposedbetween the anode and cathode, and chamber stacking means.

As will be more fully described herein below, the chamber stackcomprises a plurality of detachable sample chambers aligned along theelectrical axis between the anode and cathode. The lumens of thecoaxially aligned sample chambers are collectively capable of definingan electrically-conductive fluid column through the chamber stack.

The chamber stack further comprises a plurality of junctionalpartitions, each of the partitions positioned at a different one of thejunctions between adjacent sample chambers. The partitions prevent bulkfluid flow between the chambers that are separated by the partitions.The partitions are permeable to small ions, however, and permeable to atleast a plurality of the analytes in the sample, permitting both ion andat least some analyte flow therethrough.

The chamber stacking means is disposed completely external to thechamber stack. The stacking means facilitates the assembly of thechamber stack prior to electrophoresis and, during use, helps maintainthe fluid integrity of the chamber stack.

A schematic depiction of an embodiment of the device of the presentinvention is presented in FIG. 1.

The various components, including lid 1, are viewed from a perspectiveabove the proximal end of the partially disassembled device. As usedherein, the terms “proximal” and “distal” refer to the position of acomponent relative to the anode when the device of the present inventionis assembled and ready for use.

The embodiment shown in FIG. 1 shows seven sample chambers 2, but thenumber of sample chambers can be two, three, four, five, six, seven,eight, nine, ten or more, and still be within the scope of the presentinvention.

FIG. 2A shows several components of the device of FIG. 1 at greatermagnification. The lid is omitted for clarity.

In the embodiment shown, cathode buffer chamber 8 is integral with spilltrough 7, and anode buffer chamber 9 is integral with loading tube 4.Such integral manufacture is not required, however, and either or bothof chambers 8 and 9 may be discrete components; when discrete, either orboth of chambers 8 and 9 may be capable of resting within, or beingengaged to or engaged within, spill trough 7, or in other embodimentsconfigured to rest upon a flat surface.

In yet other embodiments, the electrical axis may be vertical duringoperation. In such embodiments, cathode buffer chamber 8 may be integralwith spill trough 7 and anode buffer chamber may be integral withloading tube 4, or either or both such chambers may be discretecomponents.

During electrophoresis, such as solution phase isoelectric focusing,sample chambers 2 are maintained in a coaxial orientation within loadingtube 4. The chambers and other components that are inserted into loadingtube 4 during electrophoresis (described in more detail below), arereferred to collectively as the chamber stack 5. Chambers 2 aretypically sealed during electrophoresis with cap seals 3. Duringelectrophoresis, chamber stack 5 is held in place within the loadingtube 4 by the screw cap 6, which applies a circumferentially uniform,proximally-directed, axial pressure on the chamber stack 5.

Once the chamber stack 5 is secured in the loading tube 4 by screw cap6, the loading tube is lowered into the spill trough 7 and the distalend of screw cap 6 sealably engaged to the cathode buffer chamber 8.

FIG. 2B shows the components of FIG. 2A fully assembled.

The axis of the electric field 12 present during electrophoresis definesthe axis along which the sample chambers are commonly aligned. The axisis defined by a line linking the submerged cathode and the submergedanode (further described below), and is shown by the bold dashed line inFIG. 2B.

FIG. 3A shows the spill trough 7 and various components of the cathoderegion of the device shown in FIG. 1.

In the embodiment shown, spill trough 7 provides a reservoir to catchany electrophoresis buffer that might leak, a useful safety feature, andalso provides underlying structural support for other components of thedevice. Spill trough 7 is optional, however, and is omitted in otherembodiments. As would be apparent, in these latter embodiments, cathodebuffer chamber 8 is designed to be a discrete component separate fromthe spill trough.

With further reference to FIG. 3A, cathode plug 15 is the componenthousing both the cathode electrode 13 and the cathode wire 14.

Cathode electrode 13 is the point of electrical contact of the devicewith the negative terminal of an external power supply (not shown).Cathode electrode 13 is in electrical contact with cathode wire 14. Inthe embodiment shown, cathode wire 14 extends from the cathode electrodethrough the interior of cathode plug 15.

In other embodiments, cathode wire 14 is routed down the outside ofcathode plug 15. Routing cathode wire 14 through the interior of cathodeplug 15 presents certain advantages, however. For example, if cathodeplug 15 is made of an insulative material, routing the conductive wireinside the insulative material reduces or eliminates off-axis linecharge, which could cause an asymmetrical field.

As shown in FIG. 3B, cathode plug 15 is engaged for use within cathodeport 16, thus bringing cathode wire 14 into contact with the interior ofbuffer chamber 8.

In some embodiments, such as those shown in FIGS. 3A and 3B, cathodeplug 15 is removably engageable with the cathode buffer chamber. Byremovably engageable is meant that the user can remove and replace it inthe cathode port without damaging the device.

In such embodiments, cathode plug 15 is inserted prior to use intocathode port 16, as shown in FIG. 3B. In one such embodiment, cathodeplug 15 simply rests in the cathode port 16. In another embodiment,cathode plug 15 snaps into the cathode port 16. In yet anotherembodiment, the cathode plug 15 screws into the cathode port 16.

In certain removable plug embodiments, cathode plug 15 is integral withor removably engaged with the lid 1, and is inserted into cathode port16 when lid 1 is engaged with cathode tab 17 and anode tab 23 (furtherdescribed below).

In alternative embodiments, cathode plug 15 is permanently orsemi-permanently sealed within the cathode port 16, and may even beintegral therewith.

The nature of the connection between the cathode plug and the cathodeport can vary but still be within the scope of the invention.

The cathode buffer chamber 8 is located at the distal end of the spilltrough 7. The cathode buffer chamber 8 is filled during operation (i.e.,during electrophoresis) with an electrically conductive cathode bufferwhich provides electrical connectivity between the cathode wire 14 andthe lumen of chamber stack 5 within the loading tube 4.

The cathode buffer chamber 8 optionally further comprises a cathodebuffer inlet 18 (see FIG. 3A) for the introduction (or removal) ofcathode buffer. The cathode buffer inlet 18 may optionally have a shapedistinguishable from the anode buffer inlet (shown and discussed herein)to ensure the user does not introduce the wrong buffer into the cathodebuffer chamber 8. In the embodiment shown in FIGS. 3A and 3B, thecathode buffer inlet 18 is shaped like a “minus sign” (−). Suchidentifying indicia may, in addition or in the alternative, be presentelsewhere on cathode buffer chamber 8.

In certain embodiments, cathode buffer chamber 8 is transparent,permitting direct visualization of dyes capable of migrating to thecathode during electrophoresis.

Cathode buffer chamber 8 may usefully be designed to have a volume of 10ml, 15 ml, even 16 ml, 17 ml, 18 ml, 19 ml, or 20 ml or more.

Spill trough 7 may also comprise a cathode tab 17 suited to fit throughthe cathode tab slot 44 in lid 1 when the device is fully assembled (seeFIGS. 9A and 9B and description below). The size and/or shape of thecathode tab 17 may optionally be distinct from the size and/or shape ofthe anode tab (discussed herein below) to ensure that the lid, and thusthe electrode cables, may only be attached with the proper polarity. Inthe embodiment shown in FIGS. 2A and 2B, cathode tab 17 is narrower(shorter) than anode tab 23.

FIG. 4A shows the loading tube and various components of the anoderegion of the device shown in FIG. 1 from a distal perspective, viewedfrom above.

Loading tube 4 holds the components of the chamber stack in coaxialalignment during assembly and operation, as discussed below withreference to FIG. 5A.

Anode plug 21 is the component housing both the anode electrode 19 andthe anode wire 20.

Anode electrode 19 is the point of electrical contact of the device withthe positive terminal of an external power supply (not shown). Anodeelectrode 19 is in electrical contact with anode wire 20. In theembodiment shown, anode wire 20 extends from the anode electrode throughthe interior of anode plug 21.

In other embodiments, anode wire 20 is routed down the outside of anodeplug 21. Routing anode wire 20 through the interior of anode plug 21presents certain advantages, however. For example, if anode plug 21 ismade of an insulative material, routing the conductive wire inside theinsulative material reduces or eliminates off-axis line charge, whichcould cause an asymmetrical field.

Anode plug 21 is engaged for use within anode port 22, thus bringinganode wire 20 into contact with the interior of anode buffer chamber 9.

In some embodiments, anode plug 21 is removably engageable with theanode buffer chamber. By removably engageable is meant that the user canremove and replace it in the anode port without damaging the device.

In such embodiments, anode plug 21 is inserted prior to use into anodeport 22, as shown in FIG. 4B. In one embodiment, anode plug 21 simplyrests in the anode port 22. In another embodiment, anode plug 21 snapsinto the anode port 22. In another embodiment, the anode plug 21 screwsinto the anode port 22.

In certain removable plug embodiments, anode plug 21 is integral with orremovably engaged with the lid 1, and is inserted into anode port 22when lid 1 is engaged with cathode tab 17 and anode tab 23.

In alternative embodiments, anode plug 21 is permanently orsemi-permanently sealed within the anode port 22, and may even beintegral therewith.

The nature of the connection between the anode plug and the anode portcan vary but still be within the scope of the invention.

Anode buffer chamber 9 may also comprise an anode tab 23 configured tofit through the anode tab slot 42 in lid 1 when the device is fullyassembled. The size and/or shape of the anode tab 23 may optionally bedistinct from the size and/or shape of the cathode tab (discussed above)to ensure that the lid, and thus the electrode cables, may only beattached with the proper polarity. In the embodiment shown in FIGS. 4Aand 4B, the anode tab 23 is wide (or long) relative to the cathode tab.

The anode buffer chamber 9 is filled during operation (i.e., duringelectrophoresis, such as IEF) with an electrically conductive anodebuffer, which provides electrical connectivity between the anode wire 20and the lumen of the chamber stack within the loading tube.

Anode buffer chamber 9 may usefully be designed to have a volume of 10ml, 15 ml, even 16 ml, 17 ml, 18 ml, 19 ml, or 20 ml or more.

The anode buffer chamber 9 optionally comprises an anode buffer inlet 24for introduction (or removal) of anode buffer. The anode buffer inlet 24may optionally have a shape distinguishable from the cathode bufferinlet 18 (shown and discussed above, FIGS. 3A and 3B) to ensure that theuser does not introduce the wrong buffer into the anode buffer chamber9. In the embodiment shown in FIGS. 4A and 4B, the anode buffer inlet 24is shaped like a “plus sign” (+).

In certain embodiments, anode buffer chamber 9 is transparent, whichpermits ready visualization of dyes that are capable of migrating to theanode during electrophoresis.

FIG. 5A shows the components of the chamber stack, partiallydisassembled, from a proximal perspective, viewed from above. FIG. 5Bshows the same components from a distal perspective, also viewed fromabove.

When fully assembled, the chamber stack comprises a plurality ofdetachable sample chambers aligned along the electrical axis between theanode and cathode; the lumens of the coaxially aligned sample arecapable collectively of defining an electrically-conductive fluid columnthrough the chamber stack.

The chamber stack further comprises a plurality of junctionalpartitions, each of the partitions positioned at a different one of thejunctions between the sample chambers. The partitions prevent bulk fluidflow between partitioned chambers, but are both ionically conductive andporous to at least a plurality of the analytes in the sample.

For use in solution phase isoelectric focusing, the partitions haveadditional features; such partitions are referred to herein asImmobilized Buffer Disks (IBD).

An IBD is a thin membrane disk containing covalently attached buffers ofdefined pH. For solution phase IEF, IBDs differing in pH are disposedbetween successive pairs of sample chambers. The IBD most proximal tothe anode is the most acidic, and the IBD closest to the cathode is themost basic. The other IBDs are arranged in decreasing order of acidityas they approach the cathodic end of series of sample chambers. In theembodiment shown in the accompanying figures, a total of six differentIBDs are used. In one embodiment, the pHs of the six IBDs, from anode tocathode, are 3.0, 4.6, 5.4, 6.2, 7.0, and 10.0.

The first six of the seven sample chambers shown in FIG. 5A, countingfrom the anode end piece, are shown preassembled. Although only theseventh chamber is shown disassembled, the junctions between all thechambers comprise the components shown explicitly for chamber seven, asdiscussed below. The various components are discussed first, by way ofexample, with respect to sample chamber seven, with reference to FIGS.6A and 6B, and then for all chambers.

Sample chambers 2 have a through bore, or lumen, that extends from theproximal face 32, closest to the anode when in the assembled chamberstack, through the distal face 34, closest to the cathode when in theassembled chamber stack, as illustrated in FIGS. 6A and 6B. FIGS. 1, 2A,5A, 6A and 6C all show the proximal face of the illustrated samplechambers, whereas FIGS. 5B and 6B show the distal faces of therespectively illustrated sample chambers.

FIG. 6A shows a sealing O-ring 29 positioned near its operationalposition at proximal face 32 of the sample chamber 2. The proximal face32 comprises seating means for sealing O-ring 29. In the embodimentsshown, the seating means include two concentric projections 33 from abase level. When assembled, the sealing O-ring 29 seats around thesecond, smaller projection, and against the first of these projections,as illustrated in FIG. 6C.

In typical embodiments, proximal projections 33 typically conformsubstantially in shape to the shape of the internal lumen of chamber 2.For example, when the lumen is circular, the proximal projections willalso typically be circular.

Also shown in FIG. 6A is a spacer 30, shown in exploded view near thedistal face 34.

The distal face 34 of chamber 2 is best illustrated in FIG. 6B. Incontrast to FIG. 6A, an IBD 28 is shown instead of a spacer 30. Intypical embodiments, spacer 30 and IBD 28 are alternatives, and only oneof the two is seated in the distal recess 36 in the distal face ofsample chambers 2. Spacer 30 will typically have outer dimensionssubstantially identical to that of IBD 28, and substantially identicalthickness. In contrast to IBD 28, however, spacer 30 will have a throughbore with shape and dimensions substantially conformal to the shape anddimensions of the chamber lumen. The positioning of a spacer betweenadjacent chambers thus serves effectively to combine the lumens of thechambers between which it is positioned into a single sample chamber ofincreased volume.

FIG. 6C shows sealing O-ring 29 assembled on proximal projections 33 ofchamber 2.

O-ring 29 acts as a face seal between adjacent chambers: upon assembly,axial compression of the chamber stack compresses O-ring 29 axially ontothe surface of either an IBD 28 or spacer 30 seated within distal recess36 of the next most proximal chamber in the stack. The O-ring holds theIBD or spacer in operational position within distal recess 36, andprevents a free electric path from becoming established around an IBDpartition.

The same O-ring contemporaneously creates a gland seal against the wallsof the distal recess of the next most proximal chamber in the stack.This aspect of the seal is not activated by axial loading, but insteadresults from the close fit between the proximal projections 33 and theouter walls of the distal recess 36 which are precisely sized toincorporate the compliant O-ring between them to form the gland seal.This gland-seal function of the single O-ring seal prevents leakage offluid between the chambers, and conversely prevents externalcontamination from entering chambers 2. Hence a single seal accomplishesboth face seal and gland seal functions.

In other embodiments, an additional O-ring may be positioned within thedistal recess of chamber 2, typically external to (i.e., distal to) theIBD or spacer. In yet other embodiments, the O-ring could be positionedfirst into the distal recess rather than the proximal projection as thetwo adjacent chambers are assembled.

The structure of the chamber stack as a whole is most easily understoodby reference to the process for its assembly. Although illustrated hereby a method of assembly, no particular order or method of assembly isrequired to create a device according to the present invention.

Anode end piece 27 is the first (that is, most proximal) componentassembled in the chamber stack (see FIG. 5A). Like chambers 2, anode endpiece 27 has a throughbore, or lumen, extending from its proximal facethrough its distal face. The lumen is typically, in both shape anddimensions, substantially identical to that of the chambers to beassembled thereto.

Also like chambers 2, the anode endpiece may include O-ring seatingmeans on its proximal face. In such case, an O-ring is applied thereto.In other embodiments, however, no O-ring seating means are provided,with seal to anode buffer chamber 9 effected, e.g., with an O-ringintegrated into the distal face of anode buffer chamber 9.

Anode endpiece 27 typically comprises a distal recess, analogous to thedistal recess on chambers 2.

A spacer 30, an IBD 28, or an alternative partition is typically placedin the anode endpiece distal recess.

In embodiments in which an IBD 28 is used, the first sample chamber maybe bounded on both sides by an IBD, and will therefore be able tocircumscribe a discrete electrophoretic fraction. When a spacer (with orwithout a nonconductive semipermeable membrane) is used, the firstsample chamber will be unable to circumscribe a discrete electrophoreticfraction.

In other embodiments, an alternative partition may be used. For example,the partition may be an ion-permeable membrane that is nonporous toanalytes above a selected molecular weight. If the molecular weightcutoff is small, the partition acts to keep analytes from traveling intothe anode buffer chamber.

Next, a first sample chamber, with O-ring assembled on the proximalprojections, is added. Alternatively, the O-ring is seated in the distalrecess of anode endpiece 27. In this latter embodiment,proximally-directed axial pressure later urges the O-ring onto theproximal projections of the first sample chamber.

The first sample chamber may include an IBD or spacer in its distalrecess at the time of its assembly to the anode end piece.Alternatively, the chamber may be assembled to the anode end piecewithout a spacer or IBD, which is thereafter placed in the distalrecess.

A second sample chamber, with O-ring seated on its proximal projections,is then added, and the process is repeated until the desired number ofsample chambers has been added. In an alternative, the O-ring is seatedin the distal recess of the first chamber, and the second chamber thenassembled thereto; proximally-directed axial pressure then urges theO-ring onto the proximal projections of the next most distal chamber.

The embodiment illustrated in FIGS. 5A and 5B includes seven totalsample chambers, although the invention also contemplates the use offewer or more chambers.

A cathode end piece 26 (FIG. 5C and in greater detail in FIG. 8) is thelast (that is, most distal) component assembled in the chamber stack(see FIG. 5A). Like chambers 2, cathode end piece 26 has a throughbore,or lumen, extending from its proximal face through its distal face. Thelumen is typically shaped and dimensioned substantially identically tothat of the chambers to be assembled thereto.

Also like chambers 2, the cathode endpiece typically includes O-ringseating means on its proximal face.

Cathode endpiece 26 typically includes a distal recess, into which aspacer, an IBD, or an alternative partition, and additionally oralternatively, an O-ring, are seated.

In the embodiments described above, junctions between sample chamberscomprise either an IBD or a spacer. A sample chamber that is not boundedat both proximal and distal faces by an IBD when assembled, but ratherby spacers, is referred to as a “blank” chamber.

Zero, one, two, three, four or more blank chambers may be used dependingon any given application of choice. For a device with “n” samplechambers, if there are fewer than “n+1” different IBD filters available,blank sample chambers are required to properly fill the loading tube.Alternatively, a user of the device may have access to n+1 IBDs but maychose not to use all of them, in which case blank sample chambers arealso used. Users of the device may opt to use fewer than all availableIBDs if the amount or number of proteins to be separated is limited, orif he or she is only interested in proteins in a specific range of pI,or for other reasons. The number of sample chambers in the IEF device,the number of blank sample chambers, the number of IBDs and their pHscan vary but still be within the scope of the present invention.

The loading tube 4 can be used to facilitate assembly of the chamberstack by holding successive components as they are added, or the chamberstack can be assembled without aid of the loading tube and subsequentlyinserted into it. FIG. 5C shows the chamber stack loaded in the loadingtube.

A removable endcap may be used to constrain the chamber stack within theloading tube.

In useful embodiments, the endcap is capable of applying a proximallydirected axial pressure to the chamber stack, urging the chambers andendpieces together, thus facilitating their sealing engagement to oneanother and to the anode and cathode buffer chambers. In particularlyuseful embodiments, the endcap is capable of applying acircumferentially uniform, proximally directed, axial pressure. FIG. 5Dshows end cap 6 engaged to stacking tube 4, constrained a chamber stackwithin the tube.

In one series of embodiments, both loading tube 4 and end cap 6 are atleast partially threaded, and the thread of the end cap is capable ofengaging the thread of the stacking tube (such embodiments of the endcap are called “screw caps” herein). In particularly useful embodiments,the stacking tube thread is external to the stacking tube, and the screwcap thread is internal to the screw cap.

The end cap has a lumen, thus permitting fluid communication between thecathode buffer chamber and the interior of the chamber stack (i.e., withlumens of the cathode end piece and the distal-most sample chamber).

In typical embodiments of the device and components of the presentinvention, sample chambers 2 include fill ports 35 (see FIGS. 6A and6B), into which cap seals 3 may be sealingly engaged.

In embodiments in which the fully engaged cap seals 3 are not flush withthe outer surface of the sample chambers, loading tube 4 may usefullyinclude an axially oriented channel 25 (see, e.g., FIGS. 4A and 4B) toaccommodate the projection of engaged cap seals 3 from the body ofsample chambers 2. In such embodiments, cap seals 3 may usefully beinserted into fill ports 35 of sample chambers 2 prior to inserting thechambers into the loading tube 4: the necessary engagement of theprojecting cap seals within (or through) the channel during loadingensures that the fill ports of all of the chambers are aligned withinthe assembled chamber stack.

Usefully, channel 25 of loading tube 4 is positioned at the top side ofloading tube 4, aligning the fill ports of chambers 2 of the chamberstack upwards (see, e.g., FIGS. 5C and 5D). This facilitates both fluidaddition and removal while the chamber stack is fully assembled.

In addition, chambers 2 may in some embodiments usefully beself-indexing—that is, configured automatically to align their fillports.

For example, the proximal projections and the distal recesses mayusefully lack rotational symmetry. In such embodiments, once theproximal projection of a second chamber is properly mated within thedistal recess of a first chamber, the two cannot rotate with respect toone another, ensuring that their fill ports are aligned. Suchself-indexing cannot be achieved by chambers that mate by screwing ontoone another, since differences in membrane thickness and tighteningtorque cause the final orientation of any fill port to be arbitrary.

In certain embodiments, the chamber lumen may advantageously conformsubstantially to such rotationally nonsymmetric shape.

Conforming the shape of the lumen to the shape of the proximalprojections and distal recess minimizes the area of the IBD outside the“active” field area, which minimizes the amount of analyte that migratesinto the IBD outside the “active” field area and is lost to thefractionation process.

Conforming the shape of the lumen to the shape of the proximalprojections also makes diffusive losses from the active to inactiveareas of the IBD uniform around the IBD circumference, by making uniformthe distribution of inactive IBD areas.

Hence, in certain useful embodiments, the chamber lumen itself followsthe shape of the seal, offset inwardly and uniformly from the internaledge of the distal recess.

The lumen of the sample chamber may also advantageously be nonsymmetricacross the horizontal plane (i.e., when the device is horizontal inuse).

In some embodiments, for example, the chamber lumen may usefully have amore acute radius of curvature at the bottom than at the top: as liquidsample is removed after fractionation, this geometry facilitates thepooling of the remaining fluid at the bottom of the chamber, thusfacilitating the complete withdrawal of the sample afterelectrophoresis. In other embodiments, the more acute radius ofcurvature is at the top: this facilitates expression of air from thechamber as the chamber is capped before electrophoresis.

FIGS. 7A and 7B present orthogonal midsectional views of an embodimentof a sample chamber of the present invention, viewed along line A-A ofFIG. 6C. FIG. 7B illustrates the operational engagement of cap seal 3within fill port 35. In the embodiment shown, the chamber lumen is arotationally nonsymmetric ovoid (pseudoellipse) (further describedbelow).

FIGS. 7C and 7D present orthogonal midsectional views of anotherembodiment, in which the fill port 35, and the corresponding portion ofthe cap seal 3, is tapered. The cap seal fits in the fill port tightlyenough to prevent sample evaporation but is readily removable by hand.FIG. 7E shows an orthogonal view of the cap seal of FIG. 7D, from thebottom. The concentric circles define the cross-sectional areas at threeplanes within the cap seal, as described in more detail below.

FIG. 9A illustrates the lid 1 for the device shown in FIG. 1. Lid 1protects the user from electrical shock and, when spill tray 7 includesdistinguishable anode and cathode tabs, helps to ensure properelectrical connection polarity.

Lid 1 optionally comprises an anode cable 41 and a cathode cable 43 thatconnect the anode electrode 19 and the cathode electrode 13 to theirrespective outlets on an external power supply (not shown) when the lidis operationally seated on the device, as shown in FIG. 9B. Theelectrode cables can be permanently attached to lid 1 or removabletherefrom.

In one embodiment, the electrode cables 41 and 43 are permanently orsemi-permanently attached to the lid such that they are not intended tobe removed by the user. In such embodiments, the electrode cables areoptionally color coded to facilitate connection with proper polarity. Insome of these embodiments, the electrodes are positioned in the lid sothat they become submerged in the anode and cathode buffer chambers whenthe lid is placed onto the apparatus, without the intermediation,respectively, of anode and cathode plugs.

In other embodiments, the electrode cables 41 and 43 are detachable fromthe lid 1. In such embodiments, holes in the lid above the anode andcathode electrodes allow the electrodes to protrude, or cables to enterthe device, to effect electrical connection. In another embodiment (notshown), prominent markings on the lid, e.g. color coded markings or“plus” and “minus” signs, may be placed on the lid at or near the anodeand cathode holes to clearly indicate to the user which power cord toattach to which electrode.

In certain embodiments, lid 1 also comprises an anode tab slot 42 and acathode tab slot 44, which optionally differ in size and/or shape. Thissize and/or shape difference makes it possible to attach the lid in onlyone orientation with respect to the electrodes, i.e. with a definedelectrical polarity.

The lid also enhances safety by preventing human contact with theelectrode or buffers during operation, since the interior of the devicecan only be accessed with the lid removed, which necessarily disconnectsthe device from the external power supply.

Lid 1 may optionally, but advantageously, be transparent, to permitvisualization of the loading tube, chamber stack, and other componentsduring electrophoresis.

To perform solution phase IEF using a device of the present invention,the chamber stack is first assembled using a plurality of IBDs, eachhaving a different, fixed, pH. A protein sample of interest isintroduced into one or more sample chambers through their respectivefill ports, and sealed without trapped air using, for example, a taperedcap seal and a tapered fill port. Any sample chambers not filled withprotein are filled with sample diluent, which will typically have a lowsalt concentration. Appropriate anode and cathode buffers are introducedthrough the respective anode and cathode buffer chamber inlets, andelectrode plugs are screwed into the electrode ports. The lid is thenput on the device, and the electrodes connected to the appropriateterminals on an external power supply.

An electric potential is applied to the device until each proteinreaches the chamber bounded by IBDs that bracket its pI.

The electrical potential is generally at least about 50 V, 100 V, 150 V,or 200V to about 1000 V, 1500 V, even as high as 2,000 V-3,000, withvalues between 50 V and 3000 V useable. The potential may be changedwithin this range during electrophoresis.

Power is turned off, the lid is taken off and samples are removed fromthe sample chambers and stored as IEF fractions defined by the IBDsbounding each Chamber.

The device and methods of the present invention do not require samplemixing or recirculation during fractionation, or means therefor. Inother embodiments, however, the entire device, such as that shown inFIG. 9B, is placed on a rocking or rotary platform duringelectrophoresis. The gentle, externally applied, motion of the samplechambers mixes samples in order, for example, to prevent precipitationof proteins at the surface of the IBD membranes and/orelectrodecantation.

External agitation of the device of the present invention is preferable,for example, to recirculation of sample through long tubing and throughperistaltic pumps, as in several prior devices. Such recirculationexposes samples to greatly increased surface area, with consequentlosses of surface-adherent components, e.g. proteins. Recirculation alsorequires that the volume of sample be increased substantially beyond thevolume of the sample chamber. Surface losses and dilution areparticularly disadvantageous with small amounts of protein.

External agitation is also superior to internal agitation using stirbars in each sample chamber. Unlike stir bar devices, the device of thecurrent invention, coupled with external agitation, does not requireintroduction of a foreign part into each sample, and does not involve aseries of fragile moving parts.

The device of the present invention enables solution phase IEFfractionation of relatively small volumes of sample, e.g. about 0.6 ml,0.7 ml, 0.8 ml, 0.9 ml, even 1.0, 1.5, 2.0, and 3.0 ml, withintermediate values permissible, such as 0.61, 0.62, 0.63, 0.64, 0.65,0.66, 0.67, 0.68, and 0.69 as exemplary volumes. Relatively small scaleprotein preparations can be used in such devices, making it possible toperform solution IEF as a prefractionation step on a number of samplesprior to analysis on 2D gels.

The device of the present invention can concentrate protein samples aswell as fractionate them.

In contrast, many of the prior devices capable of solution phase IEF aremost suited to solution IEF of preparative scale protein samples, ratherthan analytical scale samples.

Further Advantages of Component Embodiments

Electrode Plugs

Cathode plug 15, illustrated in FIGS. 3A and 3B, and anode plug 21,illustrated in FIGS. 4A and 4B, are collectively referred to herein aselectrode plugs.

As described above, the electrode plugs can usefully be removable fromtheir respective ports for ease of cleaning, repair or replacement. Inparticularly useful embodiments, the electrode plugs are identical to orotherwise interchangeable with one another: this permits the plugs to beused at either end, and permits a single spare electrode plug to serveas a replacement for either a cathode plug or an anode plug that is lostor damaged.

Typically, the electrode plugs according to the present invention aresubstantially cylindrical.

FIGS. 14A-14B show an exemplary embodiment of a cathode plug(equivalently, anode plug) in which the electrode wire advantageouslypasses through the inside of an insulative plug.

FIG. 14A is a perspective schematic view from above of such anembodiment, without the electrode wire, particularly indicating theoutlet 45 for traversal of the wire from the plug interior to itsexterior, and an optional circumferential detent 46 near the plugbottom, around which the electrode wire wraps. FIG. 14B shows the plugof FIG. 14A with the electrode wire passing from electrode, through theinsulative plug interior, out through the outlet, and around thecircumferential detent.

Circumferential positioning of the electrode wire renders the circle ofcathode wire co-planar with the circle of anode wire during use. This,in turn, ensures that the shortest distance between the anode andcathode remains the same regardless of the final rotational position ofeither plug within its respective port. This eliminates variation in thefield strength imposed by inconsistencies during assembly orre-assembly.

Recessing the wire within a circumferential detent on the electrode plugprotects the electrode wires from being bent or broken during handling,particularly cleaning. Such damage is a common problem with priordevices in which electrode wires are not fully protected. Consequently,electrode wires in the electrode plugs of the present invention need notbe as thick as electrode wires in other devices, with consequent costsavings. These advantages apply particularly to electrode wirescomprising platinum.

Loading Tube

The loading tube holds the anode end piece, sample chambers, IBDs andoptionally spacers, and cathode end piece in the proper coaxialalignment, both during assembly and in operation. As described in detailabove, the entire chamber stack can be assembled by stepwise addition ofnew components, using the loading tube to support those componentsalready assembled. Once assembled, the components are necessarilyaxially aligned along the electric field gradient.

In certain embodiments, loading tube 4 is transparent, permittingreadily visualization of the chamber stack within the tube, facilitatingcorrect assembly of the chamber stack and monitoring for problems withinthe stack, such as leakage, during electrophoresis.

Certain embodiments of the loading tube include axial channel 25,usefully positioned through the top surface of the loading tube. Thechannel accommodates cap seals that do not make a flush engagement withthe sample chambers, and positioned at the top surface of the loadingtube, usefully aligns the chamber fill ports upwardly.

Upward positioning of the fill ports allows loading and unloading of theliquid solutions with a minimum of spillage. In addition, upwardpositioning of the fill ports allows gas bubbles to float into and becaptured a region that is capable of serving as a bubble trap (seebelow). As would be readily understood, if the fill port were too farfrom vertical, i.e., on the side or at the bottom of the sample chamberthen liquid would spill out when it was opened, and gases formed duringthe electrophoresis would rise up to the top of the sample chamber andbe trapped there rather than in the bubble trap.

In certain embodiments, the anode end piece, which may lack a fill port,includes a protrusion The protrusion of the anode end piece, like theprojections of the sample chamber cap seals, is accommodated by channel25 of loading tube 4. The anode end piece protrusion is useful indisengaging all components of the chamber stack out of the loading tubeduring disassembly of the device.

End Cap

End cap 6 provides a proximally directed axial force that ensures atight seal between components of the chamber stack during assembly anduse. In some embodiments, as described above, end cap 6 is a screw cap.

Use of an end cap readily permits a circumferentially uniform axialpressure to be applied to the chamber stack; such circumferentiallyuniform pressures are difficult to achieve with prior devices thatrequire four separate nuts to be tightened onto independent alignmentrails.

In embodiments in which end cap 6 is a screw cap, engagement of thesample chamber cap seals within the loading tube channel prevents thesample chambers from rotating relative to one another and/or to theloading tube as the screw cap is tightened. Rotation of chambers and/orend pieces of the chamber stack may also be constrained by use ofself-indexing, rotationally nonsymmetric proximal projections and distalrecesses.

Tapered Cap Seal and Fill Port

FIG. 10A is a partial side midsectional view of several sample chambersassembled into a chamber stack, according to one embodiment of thepresent invention, exploded to show the cap seals positioned forinsertion. FIG. 10B shows the cap seals engaged within the samplechambers of FIG. 10A.

The fill ports provide a variety of advantages.

First, the aligned fill ports in the assembled chamber stack allows forthe loading of sample and/or buffers into the sample chambers after theapparatus is fully assembled, rather than serially during a manualassembly process, as is the case with other devices.

Second, the ports allow the fractionated samples to be extracted, e.g.with a pipette, directly from each chamber immediately afterelectrophoresis rather than, as is the case with other devices, fromindividual chambers as the device is dissembled. This reduces the risksof contamination and loss of sample due to spillage. Moreover, thecontents of each chamber can be removed in any order rather than in theorder of disassembly. For example, it may be desirable to extract theelectrophoresis sample from a particular chamber as quickly as possiblyrather than having to wait for other chambers to be removed first.

Third, the filling port creates a “bubble trap” that allows gas bubblesthat form during electrophoresis to rise and collect in a region that isoutside of the electric field. This is desirable because electriccurrent only conducts through the solution and not through the air/gasbubbles, and the analytes are present only in solution. Not only can airbubbles thus distort the electric field, they have been known to form inother electrophoretic systems and expand to the point where the gasactually occludes the fluid path to such a degree that it blocks theconduction path and therefore the entire electrophoretic process. Byallowing bubbles to collect outside of the electric field, the fillports help to maintain the field constant in cross-section and thereforeuniform in density and electromotive force (EMF) across the entire IBDsurface throughout the electrophoretic run.

In some embodiments, as illustrated in

FIGS. 7C and 7D, the walls defining fill port 35 are usefully designedso that air or other gas or supernatant fluid is expressed, rather thantrapped, as cap seal 3 is inserted into fill port 35 to seal the samplechamber. In the embodiments shown in FIGS. 7C and 7D, the tapered capseal does not fully block the fill port until the cap seal is fullyinserted, giving any air in the fill port the opportunity to escapeprior to closure.

In such embodiments, the volume of trapped air that is compressed uponfinal engagement of the cap seal within the fill port is reduced,reducing the pressure within the sample chamber and keeping the pressurebelow that which destructively displaces the gel from the IBD, or causesa fluidic leak. This feature also limits or prevents contamination ofthe fluid due to airborne substances in the sample chamber.

Alternatively, or in addition, the tapered cap seal may be used todisplace a supernatant fluid (other than air) overlaying the sample inthe sample chamber and fill port. For example, in circumstances wherethe sample volume is less than that required to completely fill thesample chamber, it may be desirable to add a supernatant fluid todisplace any air that would otherwise remain in the sample chamber. Suchfluid may be added to fill not only the sample chamber but also aportion of the fill port, to ensure that absolutely no air remains. Whenthe tapered cap seal is inserted into the fill port it will displace anyexcess supernatant fluid from the fill port (causing it to overflow outfrom the top) before finally sealing the sample chamber. The combinationof a supernatant fluid to displace air, and the tapered cap seal toallow escape of excess supernatant fluid, facilitates sealing of samplechambers without unwanted air bubbles.

Any one of a number of supernatant fluids may be used to displace airfrom sample chambers. The only requirements for the fluid are that itnot be miscible with the sample (which will typically be in aqueoussolution), that it be less dense than the sample, and that itscomponents not interact in undesirable ways with the sample. Forexample, mineral oil or 1-butanol may usefully be employed as an airdisplacing supernatant fluid.

With reference to FIG. 7D, the dimensions of the tapered region 37 of atapered cap seal may be expressed as the ratio of the cross sectionalarea of the cap seal at the top 39 of the conical region divided by thecross sectional area at the bottom 40 of the conical section. FIG. 7Epresents an orthogonal view from the bottom of the tapered cap seal. Thesmallest, inner circle in FIG. 7E represents the cross sectional area atthe bottom of the conical section, and the mid-sized circle representsthe cross sectional area at the top of the conical section. Theoutermost circle is the cross sectional area of the knob.

The taper can be expressed as the ratio of the areas of the mid-sizedand inner circles, or by the percentage by which the cross sectionalarea of the mid-size circle exceeds the cross sectional area of theinner circle. In the embodiment represented in FIG. 7E, the area of themid-sized circle is approximately 4.4 times the area of the innercircle. For this embodiment, the cross sectional area at the top of theconical region of the cap seal exceeds the cross sectional area at thebottom of the conical region by 340%. In other embodiments of thepresent invention (not shown), the cross sectional area at the top ofthe conical region of the cap seal exceeds the cross sectional area atthe bottom of the conical region only slightly, or by 20%, 50%, 100%,200%, 400% or more. The precise value of the taper may vary but still bewithin the scope of the present invention.

Immobilized Buffer Disks (IBDS)

In another aspect, the invention provides immobilized buffer disks(IBDs; synonymously, “disks”); in certain embodiments, the IBDs areparticularly adapted for use in the devices of the present invention.

As used herein, the term “disk” does not intend that the IBD necessarilypresent a circular surface as viewed along the electrical axis of thedevice; the shape of the IBD will typically conform to the shape of thesample chamber lumen, which as further described herein mayadvantageously lack rotational symmetry.

The IBDs of the present invention, when positioned as partitions betweenadjacent sample chambers in a chamber stack, are capable of interruptingbulk fluid flow through the chamber stack, but are nonetheless permeableto ions and at least a plurality of analytes desired to be analyzed. Foruse in solution phase isoelectric focusing, the disks have a fixed pH.

The IBDs comprise a porous support and a gel. In typical embodiments,the gel at least partially fills voids within the support.

In general, the support material should provide voids for gel inclusionand lack facial charges. The support can be constructed, for example,from glass fiber microfilter materials, such as Whatman GF/A, GF/B,GF/C, and GF/D filter material (Whatman Inc., Clifton, N.J., USA). Inother embodiments, the support can be constructed of polyethylene, suchas flash-spun polyethylene (Tyvek®, E.I. DuPont de Nemours, Del., USA),fritted polyethylene (Porex Corp., Fairburn, Ga., USA), sinteredpolyethylene, or bonded spun polyester fibers. In yet other embodiments,the support can include cellulose filters, cotton nonwoven fabrics, andnylon tulle fabric.

The supports of the present invention are typically thin, ranging inthickness from about 0.1 mm, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9,1.0, 1.1, 1.2, 1.3, 1.4 to about 1.5 mm, preferably from about 0.4 toabout 1.0 mm, most preferably from about 0.6 to about 0.8 mm. In oneembodiment, the supports of the present invention have a width of about0.65 mm, typically from about 0.64-0.68 mm.

Typically, the completed IBD is at most insubstantially thicker than thesupport, and the IBD thus typically ranges in thickness from about 0.1mm, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4 toabout 1.5 mm, preferably from about 0.4 to about 1.0 mm, most preferablyfrom about 0.6 to about 0.8 mm. In one embodiment, the IBDs of thepresent invention have a width of about 0.65 mm, typically from about0.64-0.68 mm.

The support is impregnated (i.e., its void volume substantially filled)with the gel material (acrylamide, agarose, etc.) of choice.

In embodiments in which the gel is a polyacrylamide gel, someformulations previously used in the art result in gel that tends to“ooze out” of the disks. In order to prepare disks having a minimalthickness that can nonetheless meet the structural requirements, theexemplary formulations set forth in Table 1, below, may be used.

TABLE 1 EXEMPLARY IBD FORMULATIONS % Crosslinker Ratio of Acrylamide asw/w % of acrylamide to Formula (w/v) acrylamide crosslinker w/w I 5 4 25II 10 4 25 III 5 8 12.5 IV 5 10 10 V 6 8 12.5 VI 6 10 17 VII 7 3 33 VIII7 4 25 IX 7 5 20 X 7 6 17 XI 7 8 12.5 XII 7 9 11 XIII 7 10 10

In an IBD of the present invention, the w/v percentage of the totalacrylamide concentration in the final gel (% T) can be as low as 4%,although % T is typically higher, such as 5%, 6%, 7%, and may be asgreat as 8%, 9%, even 10%, or more, with nonintegral values permissiblewithin the acceptable range. The percent w/w of crosslinker to totalacrylamide (% C) may be as low as 4%, and may be as high as 5%, 6%, 7%,8%, 9%, 10%, 11%, 12%, 13%, 14% even as high as 15%, with nonintegralvalues permissible within this range.

When used for solution phase isoelectric focusing, the IBDs will have afixed pH, and the gel of the IBD will typically further comprise atleast one species of copolymerized pH-conferring monomer.

In one series of embodiments, the copolymerized pH-conferring monomer isan acrylamido buffer monomer. As is known in the art, acrylamido buffersare non-amphoteric weak acids and bases having a vinyl moiety forincorporation into the gel matrix.

Acrylamido buffer monomers useful in the IBDs of the present inventionare known in the art. A number of commercially available acrylamidobuffers (Amersham Biosciences, Piscataway, N.J., USA, and Sigma-Aldrich,St. Louis, Mo., USA) include: 2-acrylamido-2-methylpropane sulfonicacid; 2-acrylamidoglycolic acid; N-acryloylglycine; 4-acrylamidobutyricacid; 2-morpholinoethylacrylamide; 3-morpholinopropylacrylamide;N,N-dimethylaminoethylacrylamide;N,N-dimethylaminoethylpropylacrylamide; andN,N-diethylaminopropylacrylamide.

In some embodiments, the gel will include a copolymer of acrylamide,N,N′-methylene-bis-acrylamide, and at least one species of acrylamidobuffer monomer.

In other embodiments, the copolymerized pH-conferring monomer is adicarboxylic acid.

The dicarboxylic acid may usefully have the formula:

in which R is selected from the group consisting of: —H, —OH, —CH₂OH,—CO₂H, —NHR′, —OCH₃, and —NR′R″, —Cl, —F, —I, and wherein R′ and R″ areeach independently selected from the group consisting of —CH₃, —CH₂CH₃,—CH₂OH, —CH₂CH₂OH, —CH₂CH₂CH₃.

Use of dicarboxylic acids as copolymerized pH-conferring monomerspresents significant advantages. The advantages are particularly evidentfor IBDs having pH fixed in the range of about 5.0 to about 6.0 withitaconic acid as the pH-conferring monomer.

Previously, disks and strips (such as IPG strips) in the pH range ofabout 5.0 to 6.0 have required high percentages of acrylamido buffers,for example as much as 10% v/v of gel, in order to achieve adequatebuffering strength. This is because the two acrylamido buffers thatbracket the desired pH have pKa values of about 4.6 (4-acrylamidobutyricacid) and about 6.2 (2-morpholinoethylacrylamide). The bufferingcapacity of a compound falls off logarithmically from the buffer's pKa.Thus, a fairly large amount of a base is required to titrate the pH of4-acrylamidobutyric acid from 4.6 to 5.4, and a fairly large amount ofan acid is required to titrate the pH of 2-morpholinoethylacrylamidefrom 6.2 to 5.4.

High percentages of acrylamido buffer disadvantageously lead tonon-uniform electrical fields, physical instability of the gel, andincrease cost. They also tend to disadvantageously increase thehydrophobicity of the gels, leading to protein retention. Highpercentages of acids and bases additionally cause, or at least increasethe amount or rate of, breakdown of polyacrylamide, decreasing shelflife.

In contrast, embodiments of the IBDs of the present invention thatinclude a gel that comprises a copolymerized dicarboxylic acid monomerare capable of having a desirably low “buffer ratio”. By “buffer ratio”is meant the ratio (mol/mol) of buffer (e.g., pH conferring monomer plustitrant) to gel. In certain embodiments of the IBDs of the presentinvention, the buffer ratio may be less than about 15%, less than about10%, less than about 5%, preferably less than about 1%, and mostpreferably less than about 0.1%.

By using less pH conferring monomer and titrant, dicarboxylicacid-containing gels of the present invention typically have a longershelf-life compared to other gels and systems that do not utilizeitaconic acid or other dicarboxylic acids.

In some embodiments, the IBD is stable when stored at 4° C. or atambient temperature for at least 2 weeks to about 3, 4, 5, 6, 7, or 8weeks, more preferably from about 1 month to about 2, 3, 4, 5, 6, 7, 8,9, 10, 11 or 12 months, most preferably from about 1 year to about 2 or3 years.

In certain embodiments, the dicarboxylic acid monomer may becopolymerized with an acrylamido buffer monomer, typically furthercopolymerized with acrylamide and an acrylamide crosslinker, such asN,N′-methylene-bis-acrylamide.

Although here described as a copolymerized component of IBDs of thepresent invention, dicarboxylic acids can be used as gel monomers in avariety of electrophoretic devices and methods, and are thus not limitedto the compositions and methods disclosed herein.

The IBDs can usefully further include visible indicia, which may appearon either or both sides of the disk.

The indicia can be incorporated using, for example, the methods andcompositions disclosed in co-pending and commonly owned U.S. Pat. No.6,521,111 B1, published U.S. patent application US 2003/0038030 A1, andpublished PCT patent application WO 01/77655 A1. Such indicia include,without limitation, any one or more of the pH of the IBD, themanufacturer, the date of manufacture, the lot number, a trademark, andany other distinctive or useful mark. Exemplary trademarks include,without limitation, ZOOM®, Invitrogen™, NOVEX® and the Invitrogen designtrademark (U.S. registration # 75912326).

Alternatively, the indicia can be printed directly on the disk using,e.g., ink jet printing approaches.

After polymerization, IBDs may be washed; washing may usefully reducecontaminants, such as unpolymerized monomers, buffer, or catalyst.

The IBDs may be washed, for example, in a low ionic strength solutionbuffered near neutrality. The wash solution can conveniently be based onthe low ionic strength buffers described in U.S. Pat. Nos. 5,578,180,5,922,185, 6,059,948, 6,096,182, 6,143,154, 6,162,338, the disclosuresof which are incorporated herein by reference in their entirety.

For example, the wash solution can usefully comprise BisTris((2-hydroxyethyl)iminotris(hydroxymethyl)methane), Tricine, glyceroland/or sorbitol, EDTA, sodium azide, and SB-14(3-(N,N-dimethylmyristylammonio)propanesulfonate), titrated to a neutralpH.

In addition or in the alternative, the IBDs can be washed with one ormore reducing agents, such as those included in the running buffersdescribed, e.g., in U.S. Pat. No. 5,578,180, the disclosure of which isincorporated herein by reference in its entirety. The reducing agentcan, e.g., be sodium bisulfite.

IBDs that are particularly adapted for use in the device of the presentinvention will typically conform in shape to the lumen of the samplechamber, albeit with dimensions sufficiently larger than those of thechamber lumen as to permit the IBD to seat within the distal recesswithout passing into the lumen itself. In preferred embodiments, theamount by which the IBD exceeds the chamber lumen in size will beuniform around the entirety of the IBD circumference.

As described above, the lumen of the sample chambers of the presentinvention may usefully lack rotational symmetry. And as furtherdescribed above, the lumen of the sample chambers of the presentinvention may advantageously lack symmetry across the horizontal plane.Accordingly, in certain embodiments, the IBDs of the present inventionlack rotational symmetry, and in other embodiments additionally lacksymmetry across the horizontal plane.

A variety of shapes comprising circular arcs of varying radii andseparation distances can be designed that meet such criteria, includinga variety of elliptical and pseudo-elliptical shapes, such as an ovoidshape.

In one series of embodiments, the IBD of the present invention is ovoid,as illustrated in FIG. 11A: this pseudoelliptical shape advantageouslylacks rotational symmetry and is nonsymmetric across the horizontalplane.

In one series of embodiments, the ovoid shape may be defined by thefollowing general formulae, in which the arcs and angles are identifiedin FIG. 11B:“Base” Arc:x ² +y ² =R _(B) ²|_(∠1) ^(∠2)“Side 1” Arc:(x−x′)² +y ² =R _(S) ²|_(∠2) ^(∠3)“Point” Arc:x ² +[y−(−y′)]² =R _(P) ²|_(∠3) ^(∠4)“Side 2” Arc:[x−(−x′)]² +y ² =R _(S) ²|_(∠4) ^(∠1)  (2)

-   -   Where R_(S1)=R_(S2)    -   and |−x′|=x′    -   and R_(P)<R_(B)<R_(S)

In a particular one of these embodiments, for example,R_(S1)=R_(S2)=11.89 mm; R_(P)=5.26 mm; R_(B)=6.37 mm; <1=0°; <2=180°;<3=213.76°; <4=326.24°; X′=|−X′|=5.52 mm; and (0, —Y′)=(0, −3.68 mm).This shape and size are suitable for use with a chamber having a lumenalvolume of about 650-750 μl.

Variations from this exemplary shape are within the scope of thisinvention. For example, the separation (y) of centerlines between the“base” and “point” arcs (dimension Y′ in the figures) may be as low asabout 3.0 mm, and as high as about 25.4 mm. That is, the separation canbe any value, including by way of non-limiting example, about 3, about4, about 5, about 6, about 7, about 8, about 9, about 10, about 11,about 12, about 13, about 14, about 15, about 16, about 17, about 18,about 19, about 20, about 21, about 22, about 23, about 24 or about 25mm. Fractions and sub-fractions of the preceding separation values canalso be used. For example, in the case of separations between about 3 toabout 4 mm, the separation can be about 3.0, about 3.1, about 3.2, about3.3, about 3.4, about 3.5, about 3.6, about 3.7, about 3.8, about 3.9 orabout 4.0 mm. As a further example, in the case of separations betweenabout 3.0 to about 3.1 mm, the separation can be about 3.00, about 3.01,about 3.02, about 3.03, about 3.04, about 3.05, about 3.06, about 3.07,about 3.08, about 3.09 or about 3.10 mm.

Variations from the exemplary size are also within the scope of thisinvention.

For example, the size can be scaled up or down at any factor suitablefor any chamber lumenal volume. By way of non-limiting example, forvolumes between 1,500 and 500 μl, a disk designed for a 750 μl chamberlumenal volume could be scaled up or down by a factor of 5× up to 0.3×,respectively.

Additional desirable dimensional and shape criteria optionally include:(1) minimization of the size of the disk; (2) minimization of the areaof the disk outside of the chamber lumen; (3) provision of a full andadequate seal between adjacent chambers; (4) maintenance of uniformityof an electrical field that mobilizes molecules through the disk; and(5) maintenance of a specific orientation between adjacent seals andsurfaces.

Kits

In another aspect, the invention provides kits for performing solutionphase isoelectric focusing using the device and components of thepresent invention.

In one series of embodiments, the kit provides components that can beassembled into the device of the present invention. The kit componentsmay be sufficient to assemble a complete device, optionally with spareparts, or may instead include only a subset of device components.

The kit may, for example, include one or more of a spill trough withintegral cathode buffer chamber, a loading tube with integral anodebuffer chamber, lid with electrodes, and chamber stack components. Thechamber stack components may include O-rings, sample chambers, samplechamber fill port cap seals, spacers and/or IBDs, cathode end piece, andanode end piece. The kit may also include one or more of end screw cap,and electrode plugs.

In another series of embodiments, the kit provides only disposableitems, such as spacers and/or IBDs.

For example, the kits may include only IBDs, for example a plurality ofIBDs each having the same fixed pH, such as pH 3.0, 4.6, 5.4, 6.2, 7.0,and 10.0. Each of the plurality of disks may be separately packaged, orthe disks may be physically segregated within a common package.

For example, in one embodiment, a plurality of IBDS, such as 2, 3, 4,even 5, 6, 7, 8, 9, or 10 or more IBDS, each having the same fixed pH,are physically segregated from one another within a single blister packor strip, such as those described in U.S. Pat. No. 4,691,820.

A blister pack, strip or package typically consists of two pieces: abase and a cover. The base is an injection-molded plastic that typicallyhas a bowl-shaped, or rectangular-shaped, recess for receiving andholding an IBD. The cover is a laminate material that typically consistsof a laminate of an aluminum foil and polypropylene. The sealing of thecover layer to the base portion, or its flange, can be carried out bythe action of heat or ultrasound, or by means of some other suitablebonding process. Once sealed, a series of individual sealed pockets(“blisters”) is formed, each one of which comprises an individual IBD.

Typically, each blister also comprises a hydrating and/or bufferingsolution to prevent drying of the IBD and to maintain the IBD ready foruse. The solution is conveniently a low ionic strength solution bufferednear neutrality, typically further comprising one or more agents tomaintain the suppleness of the support and one or more preservatives,such as sodium azide and/or a chelating agent, such as EDTA. Thesolution can conveniently be based on the low ionic strength buffersdescribed in U.S. Pat. Nos. 5,578,180, 5,922,185, 6,059,948, 6,096,182,6,143,154, 6,162,338, the disclosures of which are incorporated hereinby reference in their entirety.

For example, the solution can usefully comprise BisTris((2-hydroxyethyl)iminotris(hydroxymethyl)methane), Tricine, glyceroland/or sorbitol, EDTA, sodium azide, and SB-14(3-(N,N-dimethylmyristylammonio)propanesulfonate), titrated to a neutralpH.

Typically, the amount of solution in a given blister is between 0.8 to 5ml, with most between 1 and 3 ml, typically about 1 ml.

In another embodiment, each “blister” of a single blister pack or stripcomprises a single IBD, each of the IBDs in the pack or strip having adifferent fixed pH, so that one strip contains all of the requisite IBDsfor operation of the device. For example, a blister pack can contain aseries of IBDs having pH values of 3.0, 4.6, 5.4, 6.2, 7.0 and 10.0.

In yet other embodiments, the kits may include a plurality of suchcommon pH IBDS, with the plurality including IBDs of a plurality of pHs,permitting multiple operations of the device of the present invention.

In other embodiments, the kits may include, either separately or inconjunction with any of the kits above-described, any one or morereagents useful in solution phase isoelectric focusing, such as carrierampholytes, anode buffer (e.g., as a 50× concentrate), and cathodebuffer (e.g., for pH 3-10, at 10× concentrate). The kits may alsoinclude one or more reagents for solubilizing and/or denaturingproteins, such as urea, thiourea, and CHAPS(3-[(cholamidopropyl)dimethylammonio]-propanesulfonate).

The kits of the present invention may further comprise one or more setsof instruction, one or more protein standards, and/or one or morecontrol samples.

Yet other kits may commonly package a plurality of IBDS, with variousfixed pHs, with one or more immobilized pH gradient (IPG) strips havingpH range suitable for further analysis of fractions bracketed by theincluded IBD pHs.

Methods

The device and immobilized buffer disks of the present invention canreadily be used for solution phase isoelectric focusing (IEF),particularly for solution phase isoelectric focusing prefractionation ofsamples prior to further analysis, such as by 2D PAGE.

In a typical embodiment of the methods of the present invention, proteinsamples are prepared in sample buffer; the sample chambers andappropriate IBDs are assembled within the loading tube; the samples areloaded into the sample chambers; the sample chambers are capped with capseals; anode buffer is added to the anode reservoir and cathode bufferis added to the cathode buffer chamber; fractionation is performed; capseals are removed and sample fractions retrieved; and the fractions thenused for downstream analytical applications.

As would be understood, the steps as listed above, and their order, areexemplary.

In a first step of this exemplary method, protein samples are preparedin sample buffer. Preparation of samples for isoelectric focusing isknown in the art. See, e.g., Rabilloud, Proteome Research: TwoDimensional Gel Electrophoresis and Identification Tools, SpringerVerlag (2000) (ISBN: 3540657924) and Rabilloud, Methods Mol. Biol.112:9-19 (1999), the disclosures of which are incorporated herein byreference in their entireties.

As is well known, the major objectives of sample preparation are tocompletely solubilize the proteins, denature the proteins, reducedisulfide bonds, prevent protein modification, and maintain the proteinsin solution during solution phase IEF. Accordingly, the sample buffertypically contains: urea, for denaturation and solubilization, and/orthiourea; detergent, such as non-ionic or zwitterionic detergents, forsolubilization, such as CHAPS, NP-40, CAPSO, and sulfobetaines; DTT orDTE (dithioerythritol), as a reducing agent; and ampholytes, which helpsolubilize the proteins and maintain the pH gradient. Ampholytes aretypically used at concentrations of about 0.2-2% (v/v); higherconcentrations require longer focusing times.

Optionally, but preferably, the sample proteins can be reduced andalkylated by treating with DTT followed by alkylation in the presence ofN,N-dimethylacrylamide (DMA). Also optionally, particulate material canbe removed by high-speed centrifugation to reduce the chance of cloggingthe IBDs.

In embodiments of the device of the present invention in which samplechambers have volumes of about 700 μl, samples are typically thendiluted to about 0.6 mg protein/ml.

In the next step, the chambers are assembled as described herein above.

In one exemplary assembly method, O-rings are placed on the proximalprojections and cap seals within the fill ports of each of 7 exemplarysample chambers. The loading tube is held vertically and the anode endpiece inserted therein. A first (most proximal) chamber is then insertedinto the loading tube with its cap seal projecting through the loadingtube channel. A pH 3.0 IBD is placed in the first chamber's distalrecess. A second chamber is then inserted into the loading tube, and apH 4.6 IBD placed in its distal recess. The process is repeated withIBDs having pH 5.4, 6.2, 7.0 and 10.0. A final (7^(th)) sample chamberis inserted, followed by the cathode end piece. The end screw is screwedonto the loading tube to effect sealing engagement among the samplechambers within the loading tube.

As is described in greater detail above, the number of chambers, and theorder of IBDs therebetween, is not limited to this exemplary embodiment.

For example, with 7 chambers, the device may be assembled with fewerthan 6 IBDs, using spacers in lieu of one or more IBDs.

For example, for fractionating in the pH 4-5 range, the followingexemplary chamber stack order may be used (from proximal to distal):anode end piece, chamber, pH 3.0 IBD, chamber, spacer, chamber, pH 4.6IBD, chamber, pH 5.4 IBD, chamber, spacer, chamber, pH 10.0 IBD,chamber, cathode end piece.

For fractionating in the pH 5-6 range, the following exemplary chamberstack order may be used (from proximal to distal): anode end piece,chamber, pH 3.0 IBD, chamber, spacer, chamber, pH 5.4 IBD, chamber, pH6.2 IBD, chamber, spacer, chamber pH 10.0 IBD, chamber, cathode endpiece.

For fractionating in the pH 5-7 range, the following exemplary chamberstack order may be used (from proximal to distal): anode end piece,chamber, pH 3.0 IBD, chamber, spacer, chamber, pH 5.4 IBD, chamber, pH7.0 IBD, chamber, spacer, chamber, pH 10.0 IBD, chamber, cathode endpiece.

For fractionating in the pH 3-4 range, the following exemplary chamberstack order may be used (from proximal to distal): anode end piece,chamber, spacer, chamber, pH 3.0 IBD, chamber, pH 4.6 IBD, chamber,spacer, chamber, spacer, chamber, pH 10.0 IBD, chamber, cathode endpiece.

Next, the loading tube is inserted into the spill trough so that the endscrew sealingly engages the cathode buffer chamber.

Anode buffer and cathode buffer chambers are then filled with respectivebuffers.

Next, the samples are loaded into the sample chambers: cap seals areremoved, and sample added to each nonblank chamber (i.e., chamberpartitioned on both sides by an IBD). In one exemplary embodiment, 670μl is added to each nonblank chamber. Cap seals are reinserted into thechamber fill ports.

The lid is then engaged to the spill trough, and the electrodes attachedto a power supply.

Exemplary electrical parameters are 100 V for 20 minutes, 200 V for 80minutes, and 600 V for 80 minutes. If the power supply has a current andpower limiting capability, the current limit may usefully be set at 2 mAand the power limit at 2 W.

If current is flowing through the system, bromophenol blue included inthe sample migrates towards the anode reservoir, usefully coloring ityellow as a visual check.

Following electrophoresis, the power supply is turned off, the lidremoved, the cap seals removed and sample fractions retrieved. Thefractions may usefully be removed using, e.g., a 1 ml pipette tip on apipettor. The fractions may usefully be transferred to separatemicrocentrifuge tubes. To recover all of the fraction, the chamber maybe washed with a wash buffer (e.g., sample buffer without anyinhibitors).

The fractions may then be used for downstream analytical applications.

For example, after suitable dilution and/or desalting, the fractions maybe subjected to one dimensional electrophoresis using SDS-PAGE, or 2Dliquid chromatography/mass spectrometry (or 2D LC/MS/MS) analysis.

Alternatively, the fractions may be applied directly to immobilized pHgradient (IPG) strips for 2D PAGE analysis. Typically, neither bufferexchange nor further sample processing is required prior to IPG IEF,since the fractionated sample is in the same buffer required for firstdimension IEF using IPG strips.

In one approach, fractions are applied to IPG strips which have pH rangeabout 0.1 pH unit wider than the nominal pH range of the solution phaseIEF fraction.

The device and methods of the present invention permit fractionation ofcomplex samples by solution phase isoelectric focusing. By so doing, thedevice and methods of the present invention allow the loading ofincreased amounts of protein in downstream applications, such as2D-PAGE, reduce sample complexity, result in high resolution andidentification of low abundance proteins, increase the dynamic range ofdetection by increasing the concentration of protein species, and reduceprecipitation/aggregation artifacts of samples at high protein loadsduring 2D gel electrophoresis.

Additional Applications

Although features and aspects of the device of the present invention areillustrated above in embodiments of solution IEF devices, one of skillin the art would recognize that many aspects described would beadvantageous in the field of electrophoresis generally.

For example, the use of a loading tube sealed at the end with an endcap, such as a screw cap, that provides a circumferentially uniform,axially-directed pressure, would also be useful in sealing chambers inan electroelution device, or in a tube gel electrophoresis device.

As another example, the anode and cathode plugs described above would beuseful in any electrophoresis device. The ease of cleaning andreplacement, the sturdiness, and the ability to use very thin fragilewire would be advantageous in all forms of electrophoresis.

As yet a further example, one of skill in the art would recognize thatthe tapered fill port and cap seal arrangement described above would begenerally useful in any device in which a chamber is desirably to besealed without trapping air. Any device in which air bubbles disrupt anelectric field, such as electroelution chambers, free solutionelectrophoresis devices, and others would benefit from a sealingmechanism that helps exclude air.

The following examples are offered by way of illustration, not by way oflimitation.

EXAMPLE 1 Fractionation of Rat Liver Lysate

Rat liver tissue is lysed by sonication at a final concentration of 5%(w/v) in 7M urea, 2M thiourea, 4% CHAPS (collectively, “UTC”) andprotease inhibitors. After reduction, alkylation, centrifugation, anddetermination of the protein concentration of the supernatant fraction,samples are diluted to 0.6 mg/ml protein in UTC containing 1% ZOOM®ampholytes, pH 3-10 (Invitrogen Corp., Carlsbad, Calif., USA), 20 mMDTT, and a trace of bromophenol blue dye.

An aliquot of 3.35 ml is distributed equally into five central samplechambers of seven total chambers, each with capacity of about 670 μl,designed and assembled according to the device of the present invention.The five chambers are partitioned from one another by IBDs having pH3.0, 4.6, 5.4, 6.2, 7.0, and 10.0.

After fractionation for 3 hours, the resulting fractions are collectedand a 155 μl aliquot of each fraction is loaded onto a separate ZOOM®IPG strips (Invitrogen Corp., Carlsbad, Calif., USA). As a control, analiquot of 155 μl of unfractionated sample (92 μg unfractionated ratliver lysate proteins) is loaded directly onto a ZOOM® 3-10 NL Strip(Invitrogen Corp., Carlsbad, Calif., USA).

The ZOOM® Strips are allowed to rehydrate with the applied samplesovernight, and then focused in a ZOOM® IPGRunner™ System (InvitrogenCorp., Carlsbad, Calif., USA). The focused ZOOM strips are then appliedto NuPAGE® Novex 4-12% Bis-Tris ZOOM® gels. The resulting 2DE gels arestained with SimplyBlue™ SafeStain (Invitrogen Corp., Carlsbad, Calif.,USA) and scanned.

FIGS. 12A-12F are scanned images of the resulting 2D gels.

The pH range of the immobilized pH gradient (IPG) strip is shownimmediately beneath each gel image.

FIG. 12A is obtained with unfractionated lysate. Each of FIGS. 12B-12Fis obtained using a fraction from a different one of the device samplechambers; the pH range of the device sample chamber is shown in largetype below the IPG strip pH range.

The results show that the device of the present invention canefficiently separate a complex proteome, such as rat liver tissue, intofive well defined fractions based on pH. The fractionation reduces thesample's complexity while increasing the concentration of thefractionated proteins.

EXAMPLE 2 Improvement in Detection of Low Abundance Proteins

Rat liver lysate is prepared and fractionated in a device of the presentinvention, essentially according to Example 1.

Separate 155 μl aliquots of the pH 4.6-5.4 fraction are loadedrespectively on a pH 4.5-5.5 narrow range ZOOM® IPG strip (InvitrogenCorp., Carlsbad, Calif., USA) and a pH 4-7 ZOOM® IPG strip (InvitrogenCorp., Carlsbad, Calif., USA) and allowed to rehydrate overnight. Theapplied proteins are then focused using the ZOOM® IPGRunner™ System(Invitrogen Corp., Carlsbad, Calif., USA). The focused ZOOM® Strips areseparately applied to NuPAGE® Novex 4-12% Bis-Tris ZOOM gels. Theresulting 2DE gels are stained with SimplyBlue™ SafeStain and scanned.

Unfractionated rat liver lysate is analogously applied to a pH 4.5-5.5narrow range ZOOM® IPG strip (Invitrogen Corp., Carlsbad, Calif., USA)as a control.

FIG. 13A shows the solution phase fraction run on a pH 4-7 IPG strip,demonstrating that prefractionation in the device of the presentinvention yields a fraction with clearly defined pI range.

FIG. 13B is an equivalent solution phase pH 4.6-5.4 fraction run on a pH4.5-5.5 narrow range narrow range IPG strip. FIG. 13C shows anenlargement of the indicated region of the gel shown in FIG. 13B. Bycomparison, FIG. 13D is obtained from unfractionated rat liver lysateusing a pH 4.5-5.5 IPG strip, with FIG. 13E showing an enlargement ofthe indicated region of the gel shown in FIG. 13D.

Comparison of FIGS. 13C and 13E demonstrate that prefractionation usingthe device of the present invention improves the ability to detect lowabundance proteins.

All patents, patent publications, and other published referencesmentioned herein are hereby incorporated by reference in their entiretyas if each had been individually and specifically incorporated byreference herein.

Examples are intended to illustrate the invention and do not by theirdetails limit the scope of the claims of the invention. While preferredillustrative embodiments of the present invention are described, it willbe apparent to one skilled in the art that various changes andmodifications may be made therein without departing from the invention,and it is intended in the appended claims to cover all such deviationsand modifications that fall within the true spirit and scope of theinvention.

1. A device for solution phase electrophoretic separation of analyteswithin a sample, comprising: an anode within an anode buffer chamber; acathode within a cathode buffer chamber; a chamber stack disposedbetween said anode and said cathode; and chamber stacking means externalto said chamber stack; wherein said chamber stack comprises a pluralityof detachably mated sample chambers having lumens aligned along theelectrical axis between said anode and cathode, the lumens of saidcoaxially aligned sample chambers being collectively capable of definingan electrically conductive fluid column therethrough; wherein saidchamber stack further comprises a plurality of junctional partitions,each of said partitions positioned at a different one of the junctionsbetween adjacent sample chambers, said partitions being permeable toions and to at least a plurality of the analytes in said sample; whereinsaid chamber stacking means includes a loading tube and a removable endcap that engages said loading tube to secure said chamber stack, whereinsaid end cap comprises a lumen that permits fluid communication betweenthe interior of said chamber stack and one of said anode buffer chamberand said cathode buffer chamber.
 2. The device of claim 1, wherein thelumen of each of said sample chambers is rotationally nonsymmetricwithin the vertical plane orthogonal to the electrical axis.
 3. Thedevice of claim 1, wherein the lumen of each of said sample chambers isnonsymmetric across the horizontal plane through the midpoint of thelumen.
 4. The device of claim 3, wherein the lumen of each of saidsample chambers is ovoid within the vertical plane orthogonal to theelectrical axis.
 5. The device of claim 4, wherein the lumen of each ofsaid sample chambers has a more acute radius of curvature at the bottomthan at the top.
 6. The device of claim 1, wherein the lumen of each ofsaid sample chambers has a volume or less than about 2 ml.
 7. The deviceof claim 6, wherein-the lumen of each of said sample chambers has avolume of less than about 1.5 ml.
 8. The device of claim 7, wherein thelumen of each of said sample chambers has a volume of less than about1.0 ml.
 9. The device of claim 8, wherein the lumen of each of saidsample chambers has a volume of less than about 750 microliters.
 10. Thedevice of claim 9, wherein the lumen of each of said sample chambers hasa volume of about 600-700 microliters.
 11. The device of claim 1,wherein each sample chamber of said plurality of sample chambers furthercomprises at least one port, said at least one port capable of fluidlyconnecting the lumen of the sample chamber with the exterior of saidchamber when said chamber is mated within the chamber stack.
 12. Thedevice of claim 11, wherein the lumen of said sample chamber is ovoidwith a more acute radius of curvature at the bottom than at the top, andat least one of said sample chamber ports is at the top of each saidchamber.
 13. The device of claim 12, wherein said at least one port isinwardly tapered.
 14. The device of claim 1, wherein said samplechambers are capable of mating solely by application of axially directedforce.
 15. The device of claim 14, wherein said mated chambers areincapable of interchamber rotation.
 16. The device of claim 15, whereinthe mating of said chambers positions all said chamber ports upward. 17.The device of claim 1, wherein each said partition has the shape of asample chamber lumen.
 18. The device of claim 17, wherein each saidpartition has a rotationally nonsymmetric pseudoelliptical shape. 19.The device of claim 1, wherein at least a plurality of said partitionshave a fixed pH.
 20. The device of claim 19, wherein each of saidplurality has a different fixed pH.
 21. The device of claim 19, whereineach said partition comprises a copolymer of acrylamide, an acrylamidecrosslinker, and at least one species of pH-conferring monomer.
 22. Thedevice of claim 21, wherein said at least one species of pH-conferringmonomer is an acrylamido buffer.
 23. The device of claim 21, whereinsaid at least one species of pH-conferring monomer is a dicarboxylicacid.
 24. The device of claim 23, wherein said dicarboxylic acid isitaconic acid.
 25. The device of claim 1, wherein said chamber stackingmeans is capable of applying a circumferentially uniform, axiallydirected compressive force upon said chamber stack.
 26. The device ofclaim 1, wherein said loading tube and said removable end cap are bothat least partially threaded, and the thread of said end cap is capableof engaging the thread of said stacking tube.
 27. The device of claim26, wherein said loading tube thread is external to said tube and saidend cap thread is internal to said cap.
 28. The device of claim 1,wherein said loading tube has an axially oriented channel that extendsentirely through a wall of said tube.
 29. The device of claim 28,wherein the transaxial dimensions of said channel closely accommodatethe external projection of cap seals, said cap seals sealinglyinsertable into said sample chamber ports.
 30. The device of claim 1,wherein the lumen of said end cap permits fluid communication betweensaid anode buffer chamber and the interior of said chamber stack.